GASTRIC MUCIN SECRETION FROM CULTURED RAT EPITHELIAL-CELLS

In order to establish the measurement of gastric mucin secreted from c ultured mucous cells, rat gastric mucin was purified from secreted muc us with Sepharose CL-4B column chromatography. Gastric mucin was measu red by dot blot analysis using an enzyme-linked lectin (soybean agglut inin) assay in a good concentration-dependent manner. Surface epitheli al cells were dispersed by limited digestion of a rat everted stomach and collected by density gradient centrifugation with Percoll. These c ells were inoculated onto gelled collagen dishes, then cultured in a m edium supplemented with 10% fetal calf serum under a 5% CO2 atmosphere in air. Changing the medium after a 2-d culture, the cells were cultu red for another 3 d. During the culture, the numbers of cells each day were almost equal, but mucin contents in the cells increased, and the n dropped at day 5 after inoculation. At that time, the edge of the ce ll layer peeled off and the cells adhered to each other. Using 2-d cul tured cells, the effects of some secretagogues on mucin secretion were investigated. Carbachol, secretin, CCK-8 and prostaglandin E-2 (PGE(2 )) strongly stimulated mucin secretion, and gastrin I weakly did. Howe ver, histamine offered no stimulation.